Tumor necrosis factor (TNF) is a multifunctional cytokine that may play an important role in the pathogenesis of lung inflammation. TNF bioactivity is regulated by soluble TNF receptors that serve as TNF binding proteins. The specific aim of this project is to identify new mechanisms by which the release of soluble TNF receptors are regulated. We hypothesized that the mechanism of TNFR1 shedding might involve interactions with regulatory proteins. Utilizing a yeast two-hybrid approach, we identified ARTS-1 (Aminopeptidase Regulator of TNFR1 Shedding) as a type II integral membrane protein that associates with the TNFR1 extracellular domain. An association between membrane-associated ARTS-1 and TNFR1 was confirmed by co-immunoprecipitation experiments utilizing human pulmonary epithelial and umbilical vein endothelial cells. A direct relationship exists between membrane-associated ARTS-1 protein levels and concordant changes in the release of soluble TNFR1. Cells over-expressing ARTS-1 demonstrated increased soluble TNFR1 release and decreased membrane-associated TNFR1, while cells expressing anti-sense ARTS-1 mRNA demonstrated decreased membrane-associated ARTS-1, decreased soluble TNFR1 release and increased membrane-associated TNFR1. ARTS-1 neither bound to TNFR2 nor altered its shedding, suggesting specificity for TNFR1. Although a GST-ARTS-1 fusion protein demonstrated selective aminopeptidase activity towards non-polar amino acids, multiple lines of negative evidence suggest that ARTS-1 does not possess TNFR1 sheddase activity. These findings indicate that ARTS-1 is a multi-functional ectoprotein capable of associating with and promoting the release of soluble TNFR1. Since release of soluble TNFR1, IL-6 receptor-alpha (IL-6R), and the type II IL-1 decoy receptor (IL-1RII) can all be inhibited by hydroxamic acid based zinc metalloprotease inhibitors, we hypothesized that ARTS-1 might also regulate IL-6R and IL-1RII shedding. Reciprocal co-immunoprecipitation experiments identified that membrane-associated ARTS-1 associates with the soluble, cleaved forms of IL-6R and IL-1RII. Further, ARTS-1 promoted IL-6R and IL-1RII shedding, as demonstrated by a direct correlation between increased membrane-associated ARTS-1 protein, increased IL-6R and IL-1RII shedding, and decreased membrane-associated IL-6R and IL-1RII in cell lines overexpressing ARTS-1. Thus, ARTS-1 promotes the shedding of three distinct cytokine receptor superfamilies, the type I cytokine receptor superfamily (IL-6R), the interleukin 1/Toll-like receptor family (IL-1RII) and the TNF receptor superfamily (TNFR1). We have also identified that TNFR1 can be released from human vascular endothelial cells into the extracellular compartment as a full-length, 55-kDa receptor within the context of membranes of exosome-like vesicles. Thus, the constitutive release of TNFR1 exosome-like vesicles represents a novel pathway for the generation of soluble cytokine receptors that is distinct from the proteolytic cleavage of receptor ectodomains or the production of proteins translated from alternatively spliced mRNAs. We have recently demonstrated that proteasome inhibition induces the proteolytic cleavage and shedding of soluble TNFR1 ectodomains from human pulmonary epithelial cells, which is associated with a reduction of intracytoplasmic TNFR1 vesicles and cell surface receptors. Furtherthemore, the mechanism of TNFR1 shedding may involve the zinc metalloprotease-dependent trafficking of intracytoplasmic TNFR1 vesicles to the cell surface. These findings identify new roles for the proteasome and zinc metalloproteases in regulating the proteolytic cleavage and shedding of the TNFR1 extracellular domain. In addition, the ability of proteasome inhibition to induce the proteolytic cleavage and shedding of soluble TNFR1 ectodomains may have physiological relevance in the setting of oxidative stress, which has been associated with impaired proteasome function. The enhanced release of sTNFR1 ectodomains may also be important for patients receiving proteasome inhibitors as therapeutic agents for neoplastic disease. TNF-alpha converting enzyme (TACE, ADAM17) cleaves membrane-associated cytokines and receptors and thereby regulates inflammatory and immune events, as well as lung development and mucin production. TACE is synthesized as a latent pro-enzyme that is retained in an inactive state via an interaction between its pro-domain and catalytic domain. Although the formation of an intramolecular bond between a cysteine in the pro-domain and a zinc atom in the catalytic site had been thought to mediate this inhibitory activity, it was recently reported that the cysteine switch motif is not required. We hypothesized that the amino-terminus of the TACE pro-domain might contribute to its ability to maintain TACE in an inactive state, independent of a cysteine switch mechanism. We synthesized a 37-amino acid peptide, corresponding to TACE amino acids 18 to 54 (N-TACE18-54), and assessed whether it possessed TACE inhibitory activity. The N-TACE18-54 amino acid sequence was deduced from a RT-PCR product generated from NCI-H292 human pulmonary epithelial cell line total RNA and primers spanning the full-length TACE coding region. In an in vitro model assay system, N-TACE18-54 attenuated TACE-catalyzed cleavage of a TNFR2:Fc substrate. In addition, N-TACE18-54 inhibited constitutive TNFR2 shedding from a human monocytic cell line by 42%. A 19-amino acid, leucine-rich domain, corresponding to TACE amino acids 30 to 48, demonstrated partial inhibitory activity. Thus, we identified a sub-domain within the amino-terminus of the TACE pro-domain that attenuates TACE catalytic activity, independent of a cysteine switch mechanism, which provides new insight into the regulation of TACE enzymatic activity. We have also utilized a soluble TNF receptor (TNFR:Fc, Etanercept) in a clinical trial to demonstrate that TNF bioactivity may down-regulate allergen-mediated asthmatic airway inflammation in patients with mild-to-moderate asthma. Ongoing investigations are aimed at elucidating further the molecular mechanisms that modulate TNFR1 release to the extracellular compartment.